Histopathology in Practice

• Pathology is the study of the structural,

biochemical and functional changes in cells,
tissues and organs that underlie disease.
• The science concerned with the microscopic
structure of tissues and organs in relation to
their function.
• Also called microanatomy.
 Introduction

• Histopathological examination is used to

provide diagnostic information that is
important for timely diagnosis of disease to
determine treatment plan.

• Fresh tissue is extremely fragile & subject to

autolysis.
• Loss of specimen is a tragic result both for
patient & pathologist

• Handle all specimens with care & respect.

• Handle quickly & correctly.

• Tissue for study obtained from
– Biopsies
– Autopsies
Requirement to send specimen
• Requisition form/submission form
• Container
• Fixative
Submission form
Submission form
• One slip for one patient
• Fill properly including clinical history, pre-
operative, operative, post-operative diagnosis,
organ or tissue.
• More than one specimen for same patient.
– Label specimen name with letter A,B,C,D etc.
– e.g. A- tissue from right cheek
– B- tissue from left cheek.
Submission Form
• Help Us Help You
• ** Please provide anatomical site, lesion
description, and pertinent clinical information
on the submission form

Anatomical location, as well as critical clinical information may allow your

pathologist to provide you with the best possible diagnosis and/or
differentials
Submission Form
• If you have a list of differentials you’d like to
rule out, please mention such.

Again, please make every effort to provide necessary information in the

designated areas on biopsy submission form. It will help us help you help
your patients.
• First of all & most importantly surgeon should
Things to be taken care for during &
take adequate care to avoid contamination of
tissue withafter
tissuebiopsy surgery
from other patient.

• This may happen in operation room, clinic, or

in pathology lab.
 Specimen container

• Plastic or glass jar

• Label matching requisition slip
• Reg no.
• Full name
• Age, Sex
• Ward no, Bed no
• Site & side
• More specimen mark as A, B, C, D etc.
• Signature of doctor with date
TISSUE PROCESSING AND
PROCEDURE
 Histological Technique
• Histological technique deals with the preparation of
tissue for microscopic examination.

• The aim of good histological technique to preserve

microscopic anatomy of tissue and make them hard, so
that very thin section (4 to 5 micron) can be made.

• After staining, the section should represent the anatomy

of the tissue as close to as possible to their structure in
life.

• This is achieved by passing the total as selected part of

the tissue through a series of process
FIXATION

DEHYDRATION Tissue Processing

CLEARING
Order
INFILTRATION
IMPREGNATION

MOUNTING
EMBEDDING

TRIMMING MICROTOMY STAINING LABELING

 Types of tissue processor

• Manual tissue processor

• Automatic tissue processor

DEHYDRATION
• The process of removing
intracellular and extracellular
water.

• Dehydration should be done

rapidly without producing
considerable shrinkage or
distortion of tissues
• Tissues are dehydrated by using increasing
strength of alcohol; e.g. 50%, 70%, 90% and 100%.

• The duration for which tissues are kept in each

strength of alcohol depends upon the size of tissue,
fixative used and type of tissue.

• Delicate tissue will get high degree of shrinkage by

two great concentration of alcohol.
• The volume of alcohol should be 50-100 times that
of tissue.
• Types
1. Alcohol
i. Ethanol
ii. Methanol
iii. Isopropanol
iv. Normal and tertiary butanols
2. Glycol Ethers
i. 2-Ethoxyethanol
ii. Dioxane
iii. Polyethylene glycols(PEG)
3. Others
i. Acetone
ii. Tetrahydrofuran
iii. 2,2 dimethoxypropane(DMP) and 2,2 diethoxypropane (DEP)
iv. Phenol
 Clearing
• During dehydration water in tissue has been
replaced by alcohol.

• The next step alcohol should be replaced by

paraffin wax.

• As paraffin wax is not alcohol soluble, we

replace alcohol with a substance in which wax is
soluble. This step is call clearing.
Criteria for choosing suitable clearing agent

• Rapid removal of dehydrating agent

• Ease of removal by melted paraffin
• Minimal tissue damage
• Flammability
• Toxicity
• Cost
 Clearing of tissue is achieved by any of the following reagents:

• Xylene
• Chloroform
• Benzene
• Carbon tetrachloride
• Toluene

Note:
• Xylene is commonly used.
• Small piece of tissue are cleaned in 0.5 – 1 hour
• Larger (5cm or more thick) are cleaned in 2-4 hours.
 INFILTRATION/IMPREGNATION
• The wax is infiltrated in the interices of the tissue which
increases the optical differentiation & hardens the tissue & helps in
easy sectioning of the tissue
• In this the tissue is kept in a wax bath containing molten paraffin wax for 6 – 8
hours

Ø The various waxes which are used are,

1. Paraffin wax
2. Paraplast
3. Paraplast plus
4. Gelatin
5. Celloidin
Ideally an infiltrating and embedding medium should be:
•Soluble in processing fluids
•Suitable for sectioning and ribboning
•Molten between 30°C and 60°C
•Translucent or transparent; colorless
•Stable
•Homogeneous
•Capable of flattening after ribboning
•Non-toxic
•Odorless
•Easy to handle
•Inexpensive
 Embedding :
Ø It is done by transfering the tissue which has been
cleared of the alcohol to a mould filled with molten
wax & is allowed to cool & solidify.

Ø After solidification, a wax block is obtained which is

then sectioned to obtain ribbons.
Types of Moulds:

A. Leuckhart’s Moulds:
L- shaped brass pieces which is placed in opposing positions & can be
manipulated to increase or decrease the size of the block to be
prepared.

B. Glass or Metal petri dishes

C. Watch glass

D. Paper boats .
Leuckhart’s moulds :
• Paraffin block
The advantage of using an embedding
system are

• Ease of use
• Speed
• Tissue and holder are firmly attached,
creating a single unit
• Blocks filled immediately after sectioning
• Permanent identification
 TIME SCHEDULE (Fixation to Embedding)
FIXATION
10% Buffered Formalin 24 hours
DEHYDRATION
70% Alcohol 30 min
95% Alcohol 30 min
95% Alcohol 40 min
100% Alcohol 40 min

100% Alcohol 40 min

CLEARING
Xylene or Toluene 40 min

Xylene or Toluene 40 min

IMPREGNATION
4 Changes of Paraffin Wax 15 minutes each (1 hour)
EMBEDDING
Paraffin Wax 3 hours
TRIMMING

• The process by which when

the wax has solidified, it is
removed from the mold,
and the excess wax is cut
from the block to expose
the tissue surface in
preparation for the actual
cutting
 Section Cutting :

Ø It is the procedure in which the blocks which have been prepared are
cut or sectioned and thin strips of varying thickness are prepared.

Ø The instrument by which this is done is called as a Microtome.

v TYPES OF MICROTOMES:

• Sliding
• Rotary
• Rocking
• Freezing
• Base sledge
 MICROTOMY

• Microtomy or SECTIONING is a process whereby tissues are cut

into uniformly “thin” slices or sections with the aid of a
machine called a MICROTOME.
STAINING
• Renders the different tissue
constituents more visible,
through variations of color,
thereby promoting easier optical
differentiation of the cell and
tissue components.

• The most commonly employed

stains are EOSIN and
HEMATOXYLIN.
Procedure :
1. Deparaffinization with xylene.
2. Hydration
3. Wash under water
4. Stain with Haematoxylin for 15 min
5. Wash with water
6. Differentiate with 1 % acid alcohol
7. Wash with water for 10 min
8. Stain with 1% Eosin for 2 min
9. Wash with water.
10. Dehydration
11. Clearing with xylene
12. Dry
13. Mount
• Result :

The nucleus stains Blue

The cytoplasm stains pink.

 Mounting:
Adhesives used for fixing the sections on the
slides :

üAlbumin solution ( Mayor’s egg albumin)

üStarch paste

üGelatin
• Mountants :

ü DPX ( Distrene Dibutyl phthalate Xylene ).

ü Canada Balsam

ü Colophonium resin

ü Terpene resin
 Automation:
v Automated tissue processor:

All the before mentioned procedures upto the impregnation step can be
done automatically in a single, unmanned instrument , which is the
Automated Tissue processor.

v Advantages :

Ø It provides constant agitation during every step which ensures better

fixation & processing.

Ø It reduces the work load & in turns improves the overall output of the
laboratory.
BASIC CONCEPT
 Tissue Fixation

Aim
1. Should prevent autolysis & putrefaction of the cell
2. Should penetrate evenly and rapidly
3. Should harden the tissues
4. Increase the optical density
5. Should not cause shrinkage or swelling of the cells
6. Must not react with the receptor sites & thus must not
interfere with the staining procedure
7. Must be cheap and easily available
 Fixation
• Good fixative is most important in the
production of satisfactory results in
histopathology
 Simple Fixatives

Formalin
ADVANTAGES
1. Rapid penetration
2. Easy availability & cheap
3. Does not over harden the tissue
4. Fixes lipids for frozen sections
5. Ideal for mailing
DISADVANTAGES

1. Irritant to the nose, eyes and mucous membranes

2. Formation of precipitate of paraformaldehyde which

can be prevented by adding 11- 16 % methanol

3. Formation of black formalin pigment, Acid

formaldehyde hematin
Formulae
• 10% Formalin (Unbuffered)
– 40% Formaldehyde 100 ml
– Tap Water 900 ml
Tissue Fixation
• Specimen submit in 10% Formalin
• Formalin tissue ratio 10:1
• No other fixative should be used
• Specimen should be in a container that can be
sealed & will not leak
 Submit fresh tissue Not in Formalin
• Frozen section
• Cultures
• Renal & skin tissues for immunoflurescence
• Flow cytometry
• Chromosome studies
• Electron microscopy
 Tissue Fixation
• This is an example of an
20 cm diameter mass
lesion which was fixed at
the clinic and
subsequently sent to the
lab in a plastic, labeled,
zip lock bag devoid of
any formalin.
• Incomplete parallel cuts
minimum of 2 cm apart
(bread loafing) can be
utilized to assist with
appropriate tissue fixation
for solid organ.
Large solid specimens

Be sure to avoid complete transection or too many cuts which can both result in
loss of tissue orientation
Hollow specimen like cystic cavities:
• Hollow specimen cavity either opened or filled
with formalin by syringe or catheter or packed
with gauge or cotton soaked in formalin.

• Cystic lesions are injected with formalin after

removal of original fluid.
 Tissue Fixation
• Large specimen that
floats on fixative
should be covered by
a thick layer of
gauze.

When tissue float

• Large, flat, heavy
specimen that rest on
bottom of the
containers, the gauze
should be placed
between the container
bottom and specimen.
Large flat tissue
• Large samples can be
held to fix (at least 24
hrs) at your clinic prior to
submitting to the lab to
help avoid shipping large
volumes of formalin
which may be costly and
hazardous
• The container should be large enough to
accommodate the specimen and filled with
enough formalin to completely cover &
surround the specimen.

• The specimen should be float freely in the

container for adequate fixation.
Submitting specimens
• Submit whole specimen in a single laboratory
• Don’t divide specimen to submit in different
laboratories

Material on which diagnosis

is made (slides, blocks) can
be stored for long time &
can be evaluated by
different observers or by the
same observer at different
time.

Lobular carcinoma in fibroadenoma

Submitting specimens
• Don’t discard any tissue removed from the
body
• Submit for histopathology

Apparently innocent looking tissue may contain ugly behavior

(malignancy)
• Container should have large
enough opening
Packaging
• Fresh tissue is malleable and can
manipulate to fit into container

• Upon fixation tissue becomes rigid

and can not remove easily without
cutting or breaking the container.
No
 Packaging
• Formalin filled jars containing specimens should be
placed in a plastic bag, box, or other container with
absorbent material to absorb any leakage

The container should be

couriered or brought to the
laboratory in a biohazard bag
with a completed requisition

YES
• Paperwork should Packaging
be
placed in a separate
plastic bag to avoid
contact with formalin if
leaking does occur.

• Such contact can result

in altered and illegible
paperwork.
 Submitting Multiple Sites
• Submit multiple specimen of same patient in
multiple separate appropriately labeled jar.
 Submitting Multiple Sites

If multiple specimens are submitted in a single

container (which is less ideal) there needs to
be some method of tissue identification (i.e.
suture) to denote respective anatomical sites.
 Endoscopic Biopsies

All fragments submit in a same container

 Endoscopic Biopsies

• Do not submit endoscopic biopsies wrapped in

gauze.
• Specimens may become lost or may be crushed
during the attempted retrieval process
 Dissecting props
• Cutting board
• Forceps
• Ruler
• Scalpel / knives / saw
• Inks / dyes
• Cassettes / lids
• Biopsy pads / tissue
• Filter bags
• Weighing scales
 Specimen Categories
A Specimens only requiring transfer from container to tissue cassette.

B Specimens requiring transfer but with standard sampling, counting,

weighing or slicing.

C Simple dissection required with sampling needing a low level of

diagnostic assessment and/or preparation.

D Dissection and sampling required needing a moderate level of

assessment.

E Specimens requiring complex dissection and sampling methods

 Specimen Categories
A Specimens only requiring transfer from container to tissue
cassette.

B Specimens requiring transfer but with standard sampling, counting,

weighing or slicing.

C Simple dissection required with sampling needing a low level of

diagnostic assessment and/or preparation.

D Dissection and sampling required needing a moderate level of

assessment.

E Specimens requiring complex dissection and sampling methods

Category A Specimens
 Specimen Categories
A Specimens only requiring transfer from container to tissue cassette.

B Specimens requiring transfer but with standard sampling,

counting, weighing or slicing.

C Simple dissection required with sampling needing a low level of

diagnostic assessment and/or preparation.

D Dissection and sampling required needing a moderate level of

assessment.

E Specimens requiring complex dissection and sampling methods

Category B Specimens
 Specimen Categories
A Specimens only requiring transfer from container to tissue cassette.

B Specimens requiring transfer but with standard sampling, counting,

weighing or slicing.

C Simple dissection required with sampling needing a low level of

diagnostic assessment and/or preparation.

D Dissection and sampling required needing a moderate level of

assessment.

E Specimens requiring complex dissection and sampling methods

 Category C Specimens
Prepuce Gallbladder

Haemorrhoids

Appendix
 Specimen Categories
A Specimens only requiring transfer from container to tissue cassette.

B Specimens requiring transfer but with standard sampling, counting,

weighing or slicing.

C Simple dissection required with sampling needing a low level of

diagnostic assessment and/or preparation.

D Dissection and sampling required needing a moderate level of

assessment.

E Specimens requiring complex dissection and sampling methods

 Category D Specimens
Pigmented skin Large Intestine
lesions (Crohn’s)

Skin with markers Salivary gland tumour

 Specimen Categories
A Specimens only requiring transfer from container to tissue cassette.

B Specimens requiring transfer but with standard sampling, counting,

weighing or slicing.

C Simple dissection required with sampling needing a low level of

diagnostic assessment and/or preparation.

D Dissection and sampling required needing a moderate level of

assessment.

E Specimens requiring complex dissection and sampling methods

 Category E Specimens
Thyroid (medullary Ca) Breast cancer

Testis (seminoma) Uterus (endomet. Ca)

 What does the pathologist need to
Provide good descriptions know?
- say what you see!!
• Shape
• Colour
• Texture
• Dimensions
• Weight
• Distances from margin(s)
• Orientation markers
• Cut surface appearance etc....
• Keep your fingers crossed for good clinical history
Simple transfer
 Specimen Sampling
• Literally, taking a sample of the tissue
• Representative
• Generally, fewer blocks required if the tissue
looks uniform throughout (for benign cases)
• Sampling “rules”
 Sampling rules:
prostate chips Prostate chips (19g)

• If the chippings weigh 12g or less,

the entire specimen must be
processed

• If the chippings weigh more than

12g, a minimum of 6 cassettes
must be processed
• For every 5g over 12g, one more
cassette must be processed
Sampled in 8 cassettes:
First 12g = 6 cassettes
19g = 7g over 12g
1 cassette per 5g over = 2
more cassettes.
Simple dissection
• Specimen:
Skin from back
• Clinical details:
Sebaceous cyst
• Resection margins Inking
• Embedding instructions
• Orientation
• Distinguish between samples
• Identify the cut surface
• Acetic Acid
 Inking resection margins
Pigmented lesion

Cervical cone
 Orientation - which way up?
Anatomical
Cassette sizes
 Calcified / firm tissue

• Femoral head
• Bone Marrow Trephine
• Ethmoid mucosa / nasal polyps (cartilage)
• Nail
• Hardened cysts
• Softening

• For bony / hard tissue:

– 10% Formic Acid

• For nail:
– Phenol or hair removal
cream.
 Firm tissue testing methods
• X-ray - Expensive / ? bench space, but very
accurate

• Chemical end-point test (Ammonium

Hydroxide/Ammonium Oxalate) – very time
consuming, but accurate

• Physical manipulation - not very accurate, may

damage the specimen, but simple and
inexpensive
 Cutting instructions
• Levels
• Special stains
• Unstained Sections
• Serial sections
• Alopecia protocol
• MM sentinel lymph node
• Hirschprung’s protocol
 Specimen
Storage
• Ventilated storage units
• Largest buckets lower
shelves
• Units are in date (week)
order
• 5-weeks’ worth of storage
• Only authorised
specimens are discarded
after 5 weeks
• Any outstanding cases are
stored separately until
further notice
Processing
Embedding tools
Embedding process
• Always keep your eye on the tissue:
even for the most careful embedders,
tissue can ping like tiddlywinks. Make
sure you see where it lands!!
• Ink dots: usually used to instruct the
embedder to embed the tissue a
specific way. Make sure you know your
own lab’s protocol.

• Hide-and-seek: open lids / sponges

carefully - tissue often sticks to them. ~
why it’s important for the embedder to
know number of bits in the cassette

• Cleanliness: always watch out for

potential carry-over!!
Final block preparation
Things to Avoid
• Please help keep our technician’s fingers safe
and DO NOT submit specimens with needles
for any reason!
 Things to Avoid
• Please do no staple or suture tissue to
cardboard.
– It can damage tissue and prevent appropriate
margin assessment
Causes of rejection of specimen
• Specimen not in formalin
• Unlabeled or improperly labeled container
• Without requisition slip or incomplete
requisition slip.